3 STORAGE 1. The reagents are stable until the indicated kit expiration date if handled and stored properly. 2. When not in use, place the bottles at 4 C. WARNINGS AND PRECAUTIONS 1. Use aseptic technique when opening and dispensing reagents. 2. In case of accidental exposure of skin, mucous membranes or eyes to R1 or R2 reagents, thoroughly wash the exposed area with water. 3. This kit is designed to work properly as provided and instructed. Additions, deletions or substitutions to the procedure or reagents are not recommended, as they may be detrimental to the assay. PROCEDURAL NOTES 1. Do not leave the reagent bottles open. Replace the caps as soon as the desired volume is removed. 2. Do not allow the capped reagent bottles to sit at room temperature for long periods of time. 3. Reagent R2, methanesulfonic acid (MSA), freezes at 19 C or colder. This reagent does not need to be refrigerated, but if it is stored with the rest of the assay kit at 2-8 C, it may easily be thawed by leaving it at room temperature for a few hours prior to use. 4. To minimize error due to handling, wipe the exterior bottom of the microplate wells with a lint-free paper towel. REAGENT PREPARATION x BHT Stock Solution: 0.5 M in Acetonitrile. 2. Reagent R1: Dilute Reagent R1 3:1 with Diluent (i.e. 12 ml R1 + 4 ml Diluent). Prepare immediately before use % HCl: 12 N HCl Do not dilute prior to use in the assay. 4. Sample Blank: 75% Acetonitrile/ 25% Diluent. Add 650 µl to a microcentrifuge tube. Skip assay steps 1 and 2. The acid addition and the incubation steps are carried out on this blank µm MDA Standard Stock: Dilute the 10 mm MDA Standard 1:500 in dh 2 O (i.e. 20 µl 10 mm MDA ml dh 2 O). Prepare immediately before use. SAMPLE PREPARATION Sample Stability Unless assayed immediately, samples should be frozen at -70 C to prevent loss of MDA and HAE (3,4) and prevent new sample oxidation. Samples should not be stored at -20 C. Once thawed from -70 C storage for assay, the sample should not be refrozen. Samples should be protected from light to avoid photoxidation. Oxidation Prevention We recommend adding BHT at a final concentration of 5 mm in the buffer prior to homogenization of tissue or cells. If no antioxidant is added, new lipid peroxidation can occur during homogenization and biased values will result (2). Plasma or Serum The amount of free MDA or HAE in normal plasma or serum is at or below the limit of detection of this assay. Lipid Peroxidase Assay Kit 3/6

4 Cell Culture (8,9) Cells cultured in serum containing medium should be washed several times to remove serum components prior to homogenization. Since MDA exists as the water-soluble enolate anion at physiological ph, much of the MDA generated from lipid peroxidation in cell culture may be in the culture medium. 1. Remove cells using a rubber policeman. Lysis buffers have a high potential of interfering in this assay. Cells should be washed well in ice-cold 20 mm PBS or Tris buffer, ph 7.4, and resuspended in the same buffer. Researchers should determine the optimal number of cells to use in this assay, but a recommended starting point is 5 x 10 7 cells per ml. 2. Lyse cells by sonication, homogenization, or freeze-thaw cycles. To prevent sample oxidation during preparation, lysis should be done in the presence of 10 µl 0.5 M BHT per 1 ml of cell homogenate. 3. After homogenization, follow steps 5-7 of the tissue homogenization procedure. Tissue Homogenates (6,7) Sample homogenates should be made as concentrated as possible. The concentration of protein in the homogenate should be determined. It is recommended that 0.2 ml of a homogenate containing mg/ml protein be assayed for initial studies in a previously untested biological sample. 1. If necessary, remove blood in situ by perfusion or in vitro by rinsing with ice-cold isotonic saline (0.9% NaCl). 2. Weigh tissue. A reasonable amount to start with is 1 g tissue per 10 ml of buffer. 3. Prepare tissue homogenate in ice-cold 20 mm PBS or Tris buffer, ph 7.4. Other buffers may be used, but the researcher should confirm non-interference in the assay by measuring the MDA and/or 4-HNE standards diluted in the chosen buffer. 4. Add 10 µl 0.5 M BHT Stock Solution per 1 ml of tissue homogenate to prevent sample oxidation. A precipitate is expected and will not affect the outcome of the assay as it is removed by centrifugation. 5. After homogenization, centrifuge at 3,000 x g and 4 C for 10 minutes to remove large particles. 6. Remove an aliquot of the sample for protein determination. 7. Freeze the sample immediately at -70 C or keep on ice prior to testing. Test 0.2 ml of the homogenate in the assay. STANDARD CURVE PREPARATION Malondialdehyde is provided as an acetal because the aldehyde itself is not stable. The acetal (TMOP) is hydrolyzed during the acid incubation step at 45 C, which will generate MDA. Please see the Reagent Preparation section for preparing the 20 µm MDA Standard Stock. Table 1: Standard Curve Preparation Standard MDA Conc. Vol. of Vol. of 20 µm (µm) dh 2 O (µl) MDA Stock (µl) S S S S S S Lipid Peroxidase Assay Kit 4/6

6 system. Reproducibility Experiments in which standard samples (0-20 μm) were assayed using the same protocol over a period of 10 days established the standard error of the measurement (SEM) at less than 5%. Limitations 1. Sucrose or fructose at concentrations of 50 mm in the sample will cause a high bias in the assay. Vitamin E, when present at 15 μm, can cause a diminution in the values obtained for 4-HNE. 2. Although the standards in this assay will usually appear blue, the samples or blanks sometimes appear another color, such as pink or green. This is due to chromophores that form other than those producing the 586 nm peak. Ordinarily, these chromophores will not interfere with the A This assay measures only free MDA or 4-HNE in samples. The conditions of the assay do not provide for liberation of MDA bound to proteins via Schiff Base. 4-HNE is sufficiently reactive that it rapidly combines with proteins in tissues, forming stable adducts that are not liberated by heating at high temperatures in acid; as a consequence, there is very little free 4-HNE in tissue (1). 4. Normal tissues have very low levels of free MDA or 4-HNE, typically pmol/mg protein (2,7). Assay of a 0.2 ml sample containing 10 mg of protein derived from normal tissue will give absorbance values at 586 nm of 0.01 or less in this assay. Caution must be taken not to interpret very low absorbance values as an accurate reflection of analyte concentrations in biological samples. 5. In setting up this assay for the first time on a particular biological sample, the kinetics of color development on the sample should be followed in comparison with that of the TMOP standard. The A 586 of the sample should reach a plateau and then remain stable. If the A 586 continues to go up after the standards have achieved a stable color, the researcher should be concerned that non-mda reactivity (interference) is occurring in the sample. 6. In setting up this assay for the first time on a particular biological sample, a wavelength scan from 450 to 700 nm should be performed on the clarified sample reaction mixture and compared to that obtained with the TMOP standard. The lack of a peak at 586 nm or lack of reasonable definition to the sample profile compared to the standard would suggest interference in the sample. 7. If no antioxidant is added to the samples during homogenization and subsequent assay, a high bias due to new sample oxidation may result. REFERENCES 1. Esterbauer, H., et al.; (1991) Free Rad. Biol. Med. 11: Botsoglou, N.A., et al.; (1994) J. Agric. Food Chem. 42: Carbonneau, M.A., et al.; (1991) Clin. Chem. 37: Bull, A.W. and Marnett, L.J.; (1985) Analyt. Biochem. 149: Liu, J., et al.; (1997) Analyt. Biochem. 245: Mattson, J.P., et al.; (2002) Pathophysiology 8: Calvo, J.R., et al.; (2001) J. Cell Biochem. 80: Janciauskiene, S. and Bo Ahren; (2000) Biochem. Biophys. Res. Com. 267: Montilla-Lopez, P., et al.; (2002) European J. Pharmacology 451: For further information about this kit, its application or the procedures in this insert, please contact the Technical Service Team at Eagle Biosciences, Inc. at or at Product Developed and Manufactured in the USA Lipid Peroxidase Assay Kit 6/6

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